The goal of the proposed work is to systematically characterize the effects of genomic organization on the ability of Escherichia coli to efficiently and effectively regulate its genes through transcriptional regulation by protein transcription factors. The fundamental mechanism through which bacteria are able to sense and adapt to their environment is through the coordinated regulation of their genes by transcription factors. In theory, the ability of these regulatory molecules to control expression may depend on the relative chromosomal distance between regulators and the targets they regulate. The resulting evolutionary pressures may explain the unusually high degree of transcription factor-target co-localization on the E. coli chromosome. The proposed approach will combine experimental and theoretical methods to quantitatively characterize and model the effects of genome organization on the effectiveness of gene regulation. By using molecular methods combined with bacterial genetics, the native arrangement of regulatory networks on the E. coli chromosome will be disrupted and reorganized and the resulting effects on the efficiency, stochasticity, and biophysics of gene regulation will be measured using state of the art single cell and single molecule techniques. The resulting data can then be directly compared to theoretical models of gene regulation to distinguish between competing theories of transcription factor target search, more fully describing the fundamental underlying mechanisms driving chromosomal organization. PUBLIC HEALTH RELEVANCE: A variety of human diseases, including birth defects, cancer, and epigenetic diseases are the result of improper gene regulation. The understanding gained through these proposed studies serve as a starting point for addressing the corresponding questions regarding gene regulation in eukaryotes, where gene regulation is far less well understood, and more complex spatial organization concerns only exacerbate the difficulties of gene regulation. By developing a more complete understanding of the mechanisms underlying gene regulation in both prokaryotes and higher organisms, we will be in a better position to exploit and manipulate these mechanisms in the treatment and prevention of genetic and microbial diseases, as well as to engineer gene pathways in order to accomplish goals that will influence public health and well-being.